Abstract In eukaryotes, DNA polymerases (Pols) ? and ? play important roles in replication, but how these Pols contribute to the replication of the leading DNA strand has remained unclear. While the widely accepted model posits that Pol? replicates the leading strand and Pol? replicates the lagging strand, we recently published evidence that Pol? replicates both the leading and lagging DNA strands. Nevertheless, important issues pertaining to their roles in replication remain to be resolved. Here we propose a number of highly innovative ideas and experimental approaches to unambiguously establish the roles of Pol? and Pol? in replication. To determine whether Pol? or Pol? replicates the leading strand, in Aim 1 we will analyze Pol?-generated errors on the two DNA strands in genes located at different chromosomal sites and genome-wide in a number of different yeast strains, and we will also examine whether Pol?-generated errors occur on the leading stand; in Aim 2 we will use mutations in the PCNA binding domain of Pol? and Pol? to determine whether Pol? plays a major role in replicating both DNA strands or whether Pol? replicates the leading strand, and we will carry out studies to analyze the genetic basis of the mutator phenotype of the pol2M-644G and the exonuclease defective pol2-4 Pol? mutant alleles; and in Aim 3, we will determine whether as indicated from our genetic studies, Pol? incorporates rNMPs on the leading strand during its roles in recombination and mismatch repair, and not during replication. Altogether, we expect that the proposed studies will resolve the outstanding issues relating to the role of Pols ? and ? in replication, and they will have important bearing on DNA replication and associated DNA repair processes and on the understanding of the roles of these Pols in genomic fidelity.